A major unsolved problem of cell biology and biochemistry is elucidation of the mechanisms of intracellular lipid transport and assembly into membranes. While considerable information has accrued to define the mechanisms of intracellular protein transport, little detailed information is available to describe lipid transport processes. Lipid transport is essential for all cell, growth, development replication and homeostasis. The long term goals of this project are to define intracellular lipid transport at the molecular level. This proposal will address the problem of lipid transport in membrane biogenesis using both genetic and biochemical methods. The primary experimental system to be used in these studies is the yeast Saccharomyces cerevisiae. In this organism it is now possible to select for mutants defective in the interorganelle transport of phosphatidylserine and phosphatidylethanolamine. The genes that correct growth defects of the mutants are identified by complementation. Using these newly developed methods, we will obtain detailed molecular information about organelle biogenesis by cloning and sequencing genes that regulate the transport of phosphatidylserine to the mitochondria and Golgi apparatus; and genes that regulate transport of phosphatidylethanolamine from the mitochondria and Golgi to the endoplasmic reticulum. The genetic approach to the problem is combined with a biochemical approach that utilizes permeabilized cells to study transport mechanisms and identify transport intermediates between organelles. The location and function of the products encoded by newly identified genes is examined in permeabilized cells using reconstitution with recombinant proteins that are produced with baculovirus vectors. These studies will provide new molecular and mechanistic information about the process of membrane biogenesis and its regulation.